The primary objective of this project is to elucidate the mechanisms by which protein synthesis in eukaryotes is regulated. Using yeast as a model eukaryotic system, we are analyzing temperature-sensitive mutants of Saccharomyces cerevisiae with altered components in the protein biosynthetic pathway. By an in vitro complementation assay with pure factor, we have characterized one such mutant to be defective in elongation factor 3. Factor (EF3) is strictly required by the yeast ribosomes. Ribosomes from other eukaryotes do not require this protein. The exact function of EF3 in protein synthesis is unknown. We have purified the normal EF3 and intend to purify the mutant protein and determine the functional properties of these proteins in natural and in synthetic message translation from studies of partial reactions. Identification of the reaction blocked by mutation may lead to an understanding of the real function of EF3 in protein synthesis. The other aspect of this project will deal with the determination of the site of mutation in EF3 in order to correlate the structural alteration to the function. For this, the gene for elongation factor 3 will be isolated either by screening the existing yeast gene bank with radiolabeled antibody directed against EF3, or by cloning DNA fragments into the temperature-sensitive EF3 mutant. The transforming DNA will be isolated by the colony hybridization method. Isolated DNA will be sequenced by the methods of Maxam and Gilbert. N- and C-terminal sequences of the intact protein and of specific tryptic/chymotryptic digested fragments will be determined and will be aligned with the DNA sequence in an attempt to determine the amino acid sequence of the normal protein and to locate the site of mutation in the altered protein. For a better understanding of the structure/function relation of the factor, known DNA sequence will be altered by site-specific mutagenesis. Physically defined altered DNA fragments will be introduced into the temperature-sensitive yeast cells in order to examine the phenotypic effect of the alteration. Our long term objective is to study biogenesis and control of this and other translational factors in yeast by similar analyses described above in order to elucidate the mechanisms by which various genetic factors regulate translation.